DESCRIPTION: Although the segregation of homologous chromosomes during the first meiotic division is usually ensured by the formation of recombinational chiasmata that physically join the homologs, nonexchange chromosomes without chiasmata can also segregate properly. This is abundantly clear during meiosis I in Drosophila males, where homologous chromosomes pair with each other to form bivalents in the complete absence of recombination. Dr. McKee's laboratory has been instrumental in defining which sequences act as pairing sites to allow recognition of homologs in Drosophila primary spermatocytes. Their results to date indicate that autosomal heterochromatin plays little if any role in pairing. There appears to be a widespread distribution of weak pairing sites throughout the autosomal euchromatin, but in addition, there are some pairing sites that are highly preferred. One of these is at the base of chromosome arm 2L, and may correspond to the histone gene repeats. On the X chromosome, most euchromatic sequences and most heterochromatic sequences are inactive in male meiotic pairing (even in the presence of X chromosomal DNA elsewhere in the genome with which it could potentially pair). Instead, pairing between the X and Y chromosomes is restricted to the array of rRNA genes within the centric heterochromatin. Recent results from the McKee lab indicate that the responsible sequences are located within the intergenic spacers that separate adjacent rDNA transcriptional units. These spacers consist of several repeats of a 240 bp sequence that contain transcription-competent promoters. Mutations in these "spacer promoters" abolish both transcription and the ability to mediate pairing. This data has suggested the working hypothesis that pairing sites consist of actively transcribed sequences or to active promoters, and that particularly strong pairing sites are those that contain a high density of transcribed genes, such as is the case for rDNA and histone gene clusters. The first major aim of the research described in this proposal is to determine in more detail what kinds of sequences can serve as pairing sites. Most of the work under this specific aim will utilize a mini-chromosome assay, in which sequences to be tested for pairing activity will be "jumped" onto the free X duplication Dp(1;f)1187. Two copies of the unaltered Dp segregate randomly from each other or from an attached X-Y in the same spermatocytes, so there are no functional male-meiotic pairing sites in this minichromosome. Sequences added to the Dp will be tested for their ability to promote either proper disjunction of two minichromosomes or the regular segregation of minichromosomes from related DNA elsewhere in the genome. Sequences to be tested by this assay include: (1) rDNA, focusing on the intergenic spacer repeats; (2) histone gene repeat units; (3) random fragments of autosomal DNA; (4) Stellate sequences; (5) arrays of promoter sequences, particularly those whose activity can be controlled; and (6) arrays of binding sites for proteins (such as those in the Polycomb complex) that are associated with promoters. In a second approach, Dr. McKee will exploit X-4 translocations and terminal 4th chromosome deletions to identify pairing sites on the small 4th chromosome. The second specific aim is to understand some of the rules governing the usage of pairing sites. First, rDNA sequences that lack pairing capacity will be placed downstream of strong promoters that themselves are also incapable of promoting pairing. These constructions will be transformed into the Drosophila genome and their pairing activity checked using both the current assay (where the transgene is placed on an X chromosome lacking the rDNA repeats, and the segregation of this chromosome from the Y is monitored) and by the minichromosome assay described above. This will test whether the inactive rDNA sequences can become activated by transcriptional read-through. In another line of investigation, Dr. McKee will ask whether there are sequences outside of the "spacer promoter" that are needed for pairing, such as the binding site for topisomerase I known to be in the intergenic spacer. Finally, he will investigate the effect of base-pair mismatches on the pairing competence of rDNA intragenic spacers. The effect of mismatches at different locations throughout the spacers should reflect the actual mechanism underlying the pairing. For example, if mismatches throughout the spacers disrupt pairing, this suggests that homologous pairing is part of the process, whereas more localized effects would indicate the involvement of site-specific recombination, mediation by interactions between DNA-bound proteins or by RNA or DNA bridges, depending on the nature and location of the critical sequences.